Retrovirus-Mediated Transfection of the Tissue-type Plasminogen Activator Gene Results in Increased Thrombolysis of Blood Clots.

ABSTRACT

Tissue-type plasminogen activator (tPA) is involved in the lysis of blood clots. In this study, we attempted to target thrombolysis and enhance blood clot lysis by generating a construct (pLEGFP-N1-tPA) to integrate tPA gene into the genome of different cell lines. pLEGFP-N1-tPA construct was generated and used to target the tPA gene in different cell lines. The thrombolytic effects mediated by the supernatant from transfected HeLa cells and Linx cells were assessed using plasma thrombus plates. Furthermore, enhanced green fluorescent protein (EGFP), which was fused to the tPA gene in the pLEGFP-N1-tPA construct, was analyzed under the fluorescent microscope to assess tPA localization. We also monitored tPA activity and expression in the transfected cell lines. As part of the study, we successfully generated the pLEGFP-N1-tPA construct. The sequence of this construct was verified and the construct was subsequently used to generate the PT67/pLEGFP-N1-tPA cell line. The pLEGFP-N1-tPA construct was also used to transfect HeLa cells and Linx cells. We observed that supernatants from transfected cells were capable of lysing thrombi. In addition, tPA activity and tPA concentration were elevated in the latter supernatants and tPA was rapidly and stably expressed in the transfected cell lines. These results reveal a potentially important thrombolytic role for tPA-targeted gene therapy following cardiac valve replacement.